Il - 1 Stimulate Fiber Ring Cell Gene Expression Profile Studies In Rats And Erk Signaling Pathway In Rats Induced By Inhibition Of Il - 1 The Effect Of Fiber Ring Cell Degeneration

Background Low back pain is a common disorder in human. Intervertebral disc degeneration (IDD) is the major cause of low back pain. Many studies supported that inflammatory factors play an important role in intervertebral disc degeneration, of which IL-1is the most important one.However, the present study about the role of IL-1in the pathogenesis of IDD is limited to few genes and proteins that we thought important, there is no study about genome-wide expression profile of IL-1activated intervertebral disc cells. Extracellular regulated kinase (ERK) is an important inflammatory pathway, previous studies have showed that ERK play a crucial role in IL-1activated chondrocytes. However, the role of ERK in IL-1stimulated annulus fibrosus cells have not been studied.Objective (1) To identify the alterations in genome-wide expression profile in rat annulus fibrosus cells stimulated by IL-1, to clarify the main biological functions and signaling pathways the differentially expressed genes involved in.(2) To clarify the effect of ERK inhibition in annulus fibrosus (AF) cells stimulated by IL-1.Methods (1) Primary culture and phenotypic identification of rat annulus fibrosus cells;(2)AF cells were stimulated by IL-1for24hours,then analyzed by affymetrix gene chip, bioinformatics methods were used to analyze the biological functions and signaling pathways the differentially expressed genes involved in;(3) Rat AF cells in monolayer culture were exposed to IL-1, with or without ERK inhibition; RNA was isolated for Real time polymerase chain reaction (RT-PCR) analysis of gene expression, conditioned media was collected and analyzed for nitrite, PGE-2and IL-6, western blot was performed to detect the changes of protein expression.Results (1) Rat AF cells cultured in monolayer were round or polygonal, the cells displayed intense toluidine blue metachromatic, collagen I and collagen II expression were positive.(2) Microarray results showed that1812genes were differentially expressed after IL-1stimulation, among the known genes,841genes were upregulated and643genes were downregulated, Upregulated genes were mainly involved in inflammatory response, immune response, oxidation reduction, activation of MAPK activity, chemotaxis; downregulated genes played role in cell adhesion, response to hypoxia,cell matrix adhesion, regulation of cell growth, ion transport; MAPK signaling pathway was the most affected pathway by upregulated genes;ECM-receptor interaction was the most affected pathway by downregulated genes; The reliability of the data obtained from the microarray was verified by performing quantitative real-time PCR on 16representative genes.(3) ERK inhibition significantly decreased the upregulation of inflammatory and catabolic genes induced by IL-1; reversed the downregulation of Aggrecan, collagen â… , collagen â…¡, collagenâ…¨, IGF-1stimulated by IL-1; ERK pathway inhibition significantly decreased the protein expression of MMP-3, MMP-13induced by IL-1;while ERK pathway inhibition significantly increased the downregulated protein expression of TIMP-1, collagen â…¡ and aggrecan induced by IL-1; ERK pathway inhibition significantly decreased the production of NO, IL-6and PGE-2in culture medium induced by IL-1.Conclusion (1) IL-1plays a wide range of biological effects on AF cells, MAPK signaling pathway is the major signaling pathway activated by IL-1in AF cells;(2) ERK pathway inhibion can effectively prevent the IL-1-induced AF cell degeneration.